Although it is well-established that many malignancies can be transplanted, there is little evidence to demonstrate that a pre-malignant disease, such as myelodysplastic syndrome, can be transplanted and subsequently undergo malignant transformation in vivo. We used NUP98-HOXD13 (NHD13) mouse bone marrow to determine whether myelodysplastic syndrome could be transferred by a long-term repopulating cell. Recipients of the NHD13 myelodysplastic syndrome bone marrow displayed all of the features typical of myelodysplastic syndrome, including peripheral blood cytopenias, dysplasia, and transformation to AML. Ineffective hematopoiesis was evident in competitive repopulation assays. When equivalent numbers of WT and NHD13 cells were transplanted, equivalent numbers of cells were detected in the bone marrow, but the proportion of NHD13 cells in the peripheral blood was markedly reduced. However, despite this ineffective hematopoiesis, the NHD13 cells inexorably overgrew the WT cells. Even when transplanted with a tenfold excess of WT cells, the NHD13 cells gradually out-competed WT cells. Limiting dilution experiments demonstrated that the frequency of the cell which could transmit myelodysplastic syndrome was 1/6,000-1/16,000 and that myelodysplastic syndrome was also transferable to secondary recipients as a pre-malignant condition. Transformation to acute myeloid leukemia (AML) in primary transplant recipients was generally delayed (46-49 weeks post transplant); however, six of ten secondary transplant recipients developed AML. Since we identified the myelodysplastic syndrome-initiating cell (M-IC) in the linneg BM population, we plan to further define the M-IC cell in terms of cell surface markers. In normal hematopoiesis, the long term hematopoietic repopulating cell is contained within the LinnegSca1+Kit+ (LSK) population. Surprisingly, the LSK and LinnegSca1-Kit+ populations are both decreased in the NHD13 mice compared to WT controls. Therefore, we plan to test three subpopulations LinnegSca-Kit+, LinnegSca-Kit- and LinnegSca+Kit+ for their ability to transfer myelodysplastic syndrome into lethally irradiated WT recipients. Preliminary results from a pilot experiment are listed below; in a separate pilot experiment, 0/4 mice transplanted with 1 x 105 LinnegSca1-Kit- cells showed engraftment at 24 weeks post transplant. The LinnegSca+Kit+ population can be further refined using SLAM markers CD48 and CD150, as the LinnegSca+Kit+CD48-CD150+ population is enriched for long term repopulating hematopoietic stem cells103. Therefore, if our preliminary data is confirmed, we will repeat the transplant experiments using LinnegSca+Kit+CD48-CD150+ cells, to determine if the M-IC resides in this long term repopulating compartment. One reason for the difficulty in developing effective treatments for myelodysplastic syndrome is that there are no myelodysplastic syndrome cell lines which can be used to model or study the disease; cell lines that are described as myelodysplastic syndrome cell lines are really AML cell lines which have evolved from patients with myelodysplastic syndrome. Although numerous investigators have attempted to develop xenograft models for myelodysplastic syndrome, these attempts have met with little success. Given that the NHD13 mice develop a highly penetrant myelodysplastic syndrome that closely resembles the human disease, we have begun studies to determine if these mice are a useful pre-clinical model for myelodysplastic syndrome. Our initial studies have used the DNA-methyltransferase inhibitor 5-azacytidine. The dose we chose was 0.1 mg via subcutaneous injection daily for 7 days; this constituted one cycle of chemotherapy. Cycles were repeated every 4 weeks. This dosing schedule closely approximates the schedule used clinically (75 mg/M2 daily for 7 days). Our pilot study included 3 groups of mice [NHD13 mice injected with 5-azacytidine (n=6), NHD13 mice injected with saline (n=4), and WT mice injected with 5-azacytidine (n=4)] for which we obtained CBCs before the first dose of 5-azacytidine and before every other cycle subsequently. After 16 weeks of therapy, the results appeared promising, as the treated NHD13 mice showed a significant increase in hemoglobin compared to the saline treated NHD13 mice (2.21 +/- 1.47 g/dL vs 0.13 +/- 0.66 g/dL, p=0.02). Unfortunately, three of the NHD13 treated mice were found dead in the following two months, and we were not able to determine the cause of death for these mice. The three remaining mice had gradual decreases in hemoglobin over the next 12 weeks, and were close to baseline hemoglobin levels. Nonetheless the observation that these mice had stable hemoglobin levels after 28 weeks was encouraging. We plan to repeat this experiment with a larger cohort of mice. Since the WT mice showed no evidence of pancytopenia, and had only one death (likely due to hemorrhage caused by the injection), we consider the 0.1 mg SQ dose to not be overly toxic. To determine whether we were achieving levels of 5-azacytidine adequate to cause cytosine demethylation, we will examine global and gene-specific methylation status. If the 5-azacytidine treated samples show evidence of cytosine demethylation, we will continue using the dose and schedule outlined above. If the samples show no evidence of cytosine demethylation, we will consider revising the dosage schedule. In addition to the experiments outlined above, we have transferred NHD13 mice to colleagues at several academic institutions, and are in the process of licensing NHD13 mice in order for them to be sent to three separate biotech companies for pre-clinical studies. These colleagues have plans to treat NHD13 mice with a variety of agents, including histone deacetylase inhibitors, apoptosis inhibitors, and angiogenesis inhibitors.